Human collagen Krox up-regulates type I collagen expression in normal and scleroderma fibroblasts through interaction with Sp1 and Sp3 transcription factors.

Despite several investigations, the transcriptional mechanisms that regulate the expression of both type I collagen genes (COL1A1 and COL1A2) in either physiological or pathological situations, such as scleroderma, are not completely known. We have investigated the role of hc-Krox transcription factor on type I collagen expression by human dermal fibroblasts. hc-Krox exerted a stimulating effect on type I collagen protein synthesis and enhanced the corresponding mRNA steady-state levels of COL1A1 and COL1A2 in foreskin fibroblasts (FF), adult normal fibroblasts (ANF), and scleroderma fibroblasts (SF). Forced hc-Krox expression was found to up-regulate COL1A1 transcription through a -112/-61-bp sequence in FF, ANF, and SF. Knockdown of hc-Krox by short interfering RNA and decoy strategies confirmed the transactivating effect of hc-Krox and decreased substantially COL1A1 transcription levels in all fibro-blast types. The -112/-61-bp sequence bound specifically hc-Krox but also Sp1 and CBF. Attempts to elucidate the potential interactions between hc-Krox, Sp1, and Sp3 revealed that all of them co-immunoprecipitate from FF cellular extracts when a c-Krox antibody was used and bind to the COL1A1 promoter in chromatin immunoprecipitation assays. Moreover, hc-Krox DNA binding activity to its COL1A1-responsive element is increased in SF, cells producing higher amounts of type I collagen compared with ANF and FF. These data suggest that the regulation of COL1A1 gene transcription in human dermal fibroblasts involves a complex machinery that implicates at least three transcription proteins, hc-Krox, Sp1, and Sp3, which could act in concert to up-regulate COL1A1 transcriptional activity and provide evidence for a pro-fibrotic role of hc-Krox

Effets des facteurs transcriptionnels hc-Krox et NF-kappaB sur la régulation de l'expression du collagène de type I dans des fibroblastes dermiques humains sains et slcérodermiques

Dans cette étude, nous avons recherché les effets des facteurs trans hc-Krox ( human c-Krox ), Sp1 ( Specific Protein-1 ), Sp3 et NF-kappaB ( Nuclear Factor-kappaB ) sur l expression du collagène I dans des fibroblastes dermiques humains normaux d adultes (FNA) et sclérodermiques (FS). Ainsi, nous avons démontré que les facteurs Sp1/3 et hc-Krox, trois facteurs à doigts à zinc, activent l expression du collagène I au niveau protéique et au niveau des taux d ARNm COL1A1 et COL1A2, dans des FNA et des FS. De plus, ces facteurs augmentent l activité transcriptionnelle du gène COL1A1 par l intermédiaire de la région promotrice proximale de 112 pb dans les deux souches fibroblastiques, via des séquences cis caractérisées fonctionnellement. A l inverse, la sous-unité p65 de NF-kappaB inhibe l activité transcriptionnelle de COL1A1 chez les FNA et les FS, via la même région promotrice ne comportant pourtant pas de séquence consensus pour ce facteur. A cet égard, nous avons montré que pour exercer ses effets, le facteur NF-kappaB interagissait in vitro et in vivo avec Sp1, Sp3 et hc-Krox, et était de ce fait recruté sur le promoteur COL1A1. Des études complémentaires dans des FNA et des FS ont montré que l expression du collagène I est corrélée avec l activité de liaison de hc-Krox sur le promoteur COL1A1. Nous avons également réalisé des expériences préliminaires sur le vieillissement cutané d individus de tranches d âges différentes qui ont montré que les expressions du collagène I et du facteur activateur hc-Krox diminuaient avec l âge ; à l inverse, l activité de liaison de NF-kappaB semble augmenter au cours du vieillissementIn this study, we have investigated the roles of transcription factors hc-Krox (human c-Krox), Sp1 (Specific Protein-1), Sp3 and NF-kappaB (Nuclear Factor-kappaB) on type I collagen expression by normal human (NHF) and scleroderma (SF) fibroblasts. Our results show that the three zinc-finger proteins hc-Krox, Sp1 and Sp3 are strong activators of type I collagen expression by increasing type I collagen protein synthesis and steady-state levels of COL1A1 and COL1A2 mRNA in NHF and SF. Moreover, they up-regulate COL1A1 gene transcriptional activity through a region localized in the 112 bp proximal promoter in which we have identified the functional cis-responsive elements. We also demonstrated that NF-kappaB down-regulates COL1A1 by a transcriptional control and through the same region by which the zinc-finger mediate their effects. Despite no existing consensus sequence for NF-kappaB in the COL1A1 proximal promoter, we find that all the trans factors, including NF-kappaB, bind to the COL1A1 promoter in chromatin immunoprecipitation assays. Furthermore, Sp1/3/hc-Krox are necessary to mediate the inhibitory effect of NF-kB on COL1A1 in NHF and SF since they are found to interact each others. Moreover, complementary experiments performed in NHF and SF demonstrated that type I collagen synthesis is correlated with the c-Krox DNA binding activity on the COL1A1 promoter. Besides, we realized preliminary experiments showing that type I collagen and c-Krox expression decrease during aging, although NF-kappaB binding activity seems to increase.CAEN-BU Sciences et STAPS (141182103) / SudocSudocFranceF

Interleukin-1 and Transforming Growth Factor-ß 1 as Crucial Factors in Osteoarthritic Cartilage Metabolism

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Chondroitin sulphate decreases collagen synthesis in normal and scleroderma fibroblasts through a Smad-independent TGF-β pathway - implication of C-Krox and Sp1

International audienceDespite several investigations, the transcriptional mechanisms which regulate the expression of both type I collagen genes (COL1A1 and COL1A2) in either physiological or pathological situations, such as scleroderma, are not completely known. In this study, we determined the effects of both native ichtyan chondroïtin sulphate (CS) and its derived hydrolytic fragments (CSf) on human normal (NF) and scleroderma (SF) fibroblasts. Here, we demonstrate for the first time that CS and CSf exert an inhibitory effect on type I collagen protein synthesis and decrease the corresponding mRNA steady-state levels of COL1A1 and COL1A2 in NF and SF. These glycosaminoglycan molecules repress COL1A1 gene transcription through a -112/-61 bp sequence upstream the start site of transcription and imply hc-Krox and Sp1 transcription factors. In addition, CS and CSf induced a down-regulation of TbetaRI expression. As a conclusion, our findings highlight a possible new role for CS and CSf as anti-fibrotic molecules and could help in elucidating the mechanisms of action by which CS and CSf exert their inhibitory effect on type I collagen synthesis

c-Krox down-regulates the expression of UDP–glucose dehydrogenase in chondrocytes

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Estrogens induce rapid cytoskeleton re-organization in human dermal fibroblasts via the non-classical receptor GPR30.

The post-menopausal decrease in estrogen circulating levels results in rapid skin deterioration pointing out to a protective effect exerted by these hormones. The identity of the skin cell type responding to estrogens is unclear as are the cellular and molecular processes they elicit. Here, we reported that lack of estrogens induces rapid re-organization of the human dermal fibroblast cytoskeleton resulting in striking cell shape change. This morphological change was accompanied by a spatial re-organization of focal adhesion and a substantial reduction of their number as evidenced by vinculin and actin co-staining. Cell morphology and cytoskeleton organization was fully restored upon 17β-estradiol (E2) addition. Treatment with specific ER antagonists and cycloheximide respectively showed that the E2 acts independently of the classical Estrogen Receptors and that cell shape change is mediated by non-genomic mechanisms. E2 treatment resulted in a rapid and transient activation of ERK1/2 but not Src or PI3K. We show that human fibroblasts express the non-classical E2 receptor GPR30 and that its agonist G-1 phenocopies the effect of E2. Inhibiting GPR30 through treatment with the G-15 antagonist or specific shRNA impaired E2 effects. Altogether, our data reveal a novel mechanism by which estrogens act on skin fibroblast by regulating cell shape through the non-classical G protein-coupled receptor GPR30 and ERK1/2 activation

Signaling pathways involved in desflurane-induced postconditioning in human atrial myocardium in vitro

BACKGROUND: Isoflurane and sevoflurane have been shown to elicit myocardial postconditioning, but the effect of desflurane remain unknown. The authors studied the mechanisms involved in desflurane-induced myocardial postconditioning. METHODS: Contracting isolated human right atrial trabeculae (34 degrees C, stimulation frequency 1 Hz) were exposed to 30-min hypoxia followed by 60-min reoxygenation. Desflurane at 3%, 6%, and 9% was administered during the first 5-min of reoxygenation. Postconditioning with 6% desflurane was studied in the presence of 1 microM calphostin C, a protein kinase C inhibitor; 800 mm 5-hydroxydecanoate, a mitochondrial adenosine triphosphate-sensitive potassium channels antagonist; 1 microM Akt inhibitor; 20 microM PD89058, an extracellular-regulated kinase 1/2 inhibitor; and 1 microM SB 202190, a p38 mitogen-activated protein kinase inhibitor. The force of contraction at the end of the 60-min reoxygenation period was compared (mean +/- SD). The p38 mitogen-activated protein kinase phosphorylation was studied using Western blotting. RESULTS: Desflurane at 3% (77 +/- 10% of baseline), 6% (90 +/- 14% of baseline), and 9% (86 +/- 11% of baseline) enhanced the recovery of force after 60 min of reoxygenation as compared with the control group (51 +/- 9% of baseline; P < 0.001). Calphostin C (55 +/- 3% of baseline), 5-hydroxydecanoate (53 +/- 3% of baseline), Akt inhibitor (57 +/- 8% of baseline), PD89058 (64 +/- 6% of baseline), and SB 202190 (61 +/- 3% of baseline) abolished desflurane-induced postconditioning. Western blot analysis showed that 6% desflurane increased p38 mitogen-activated protein kinase phosphorylation. CONCLUSIONS: In vitro, desflurane postconditioned human atrial myocardium through protein kinase C activation, opening of mitochondrial adenosine triphosphate-sensitive potassium channels, Akt and extracellular-regulated kinase 1/2 activation, and p38 mitogen-activated protein kinase phosphorylation

Interleukin-6 (IL-6) and/or Soluble IL-6 Receptor Down-regulation of Human Type II Collagen Gene Expression in Articular Chondrocytes Requires a Decrease of Sp1·Sp3 Ratio and of the Binding Activity of Both Factors to the COL2A1 Promoter

International audienceType II collagen is composed of alpha1(II) chains encoded by the COL2A1 gene. Alteration of this cartilage marker is a common feature of osteoarthritis. Interleukin-6 (IL-6) is a pro-inflammatory cytokine that needs a soluble form of receptor called sIL-6R to exert its effects in some cellular models. In that case, sIL-6R exerts agonistic action. This mechanism can make up for the partial or total absence of membrane-anchored IL-6 receptors in some cell types, such as chondrocytes. Our study shows that IL-6, sIL-6R, or both inhibit type II collagen production by rabbit articular chondrocytes through a transcriptional control. The cytokine and/or sIL-6R repress COL2A1 transcription by a -63/-35 sequence that binds Sp1.Sp3. Indeed, IL-6 and/or sIL-6R inhibit Sp1 and Sp3 expression and their binding activity to the 63-bp promoter. In chromatin immunoprecipitation experiments, IL-6.sIL-6R induced an increase in Sp3 recruitment to the detriment of Sp1. Knockdown of Sp1.Sp3 by small interference RNA and decoy strategies were found to prevent the IL-6- and/or sIL-6R-induced inhibition of COL2A1 transcription, indicating that each of these Sp proteins is required for down-regulation of the target gene and that a heterotypic Sp1.Sp3 complex is involved. Additionally, Sp1 was shown to interact with Sp3 and HDAC1. Indeed, overexpression of a full-length Sp3 cDNA blocked the Sp1 up-regulation of the 63-bp COL2A1 promoter activity, and by itself, inhibits COL2A1 transcription. We can conclude that IL-6, sIL-6R, or both in combination decrease both the Sp1.Sp3 ratio and DNA-binding activities, thus inhibiting COL2A1 transcription

NF-κB Accumulation Associated with COL1A1 Trans activators Defects during Chronological Aging Represses Type I Collagen Expression through a –112/–61-bp Region of the COL1A1 Promoter in Human Skin Fibroblasts

International audienceThe aging process, especially of the skin, is governed by changes in the epidermal, dermo-epidermal, and dermal compartments. Type I collagen, which is the major component of dermis extracellular matrix (ECM), constitutes a prime target for intrinsic and extrinsic aging-related alterations. In addition, under the aging process, pro-inflammatory signals are involved and collagens are fragmented owing to enhanced matrix metalloproteinase activities, and fibroblasts are no longer able to properly synthesize collagen fibrils. Here, we demonstrated that low levels of type I collagen detected in aged skin fibroblasts are attributable to an inhibition of COL1A1 transcription. Indeed, on one hand, we observed decreased binding activities of specific proteins 1 and 3, CCAAT-binding factor, and human collagen-Kruppel box, which are well-known COL1A1 transactivators acting through the -112/-61-bp promoter sequence. On the other hand, the aging process was accompanied by elevated amounts and binding activities of NF-kappa B (p65 and p50 subunits), together with an increased number of senescent cells. The forced expression of NF-kB performed in young fibroblasts was able to establish an old-like phenotype by repressing COL1A1 expression through the short -112/-61-bp COL1A1 promoter and by elevating the senescent cell distribution. The concomitant decrease of transactivator functions and increase of transinhibitor activity is responsible for ECM dysfunction, leading to aging/senescence in dermal fibroblasts

GPR30 mediates the effects of 17β-estradiol on hDF shape.

<p><b>a.</b> Efficiency of shRNA vectors. hDF were transfected with shGPR30 (#1 and #2) or control (shC). Expression of the indicated proteins was determined 48 h after transfection. <b>b.</b> ERK phosphorylation under the indicated conditions. Ratios of phosphorylated ERK (p-ERK) to total ERK (t-ERK) are indicated. Lower panel: quantification of the western blots performed on the four donors. Data are presented as mean +/- s.e.m. relative to vehicle-treated control. <b>c.</b> Cells shape monitored under the indicated conditions as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120672#pone.0120672.g001" target="_blank">Fig. 1b</a>. n = 400 cells. ns: not significant; ***<i>p</i><0.001.</p